Diagnostic method

ABSTRACT

It has been demonstrated that the level of HBP increases in individuals that subsequently develop severe sepsis. Accordingly, the level of HBP, HBP/WBC ratio or HBP/NC ratio in an individual can be used to determine whether or not an individual is at risk of developing severe sepsis.

FIELD OF THE INVENTION

The invention relates to the diagnosis of susceptibility to and theprevention of the development of severe sepsis.

BACKGROUND OF THE INVENTION

Sepsis is a systemic inflammatory response to infection, which causesorgan failure and death in severe cases. It is an increasingly commoncause of morbidity and mortality, particularly in elderly,immuno-compromised, and critically ill individuals. Sepsis has beenreported to be the most common cause of death in the non-coronaryintensive care unit (Bone R C et al; Chest. 1992 June; 101(6): 1644-55).It occurs in 1-2% of all hospitalizations and mortality rates range from20% for sepsis to 40% for severe sepsis to >60% for septic shock (asub-category of severe sepsis) (Leibovici; Ann Intern Med 1991;114(8):703, Martin et al; N Engl J Med. 2003 Apr. 17; 348(16):1546-54).

The clinical definition of sepsis is the presence of two or more of thefollowing conditions:

(1) fever (temperature >38° C.) or hypothermia (temperature <36° C.);

(2) heart rate >90 beats per minute;

(3) respiratory rate >20 breaths per minute or PaCO₂<32 nun Hg; and

(4) white blood cell count >12(×10⁹cells/L) or <4(×10⁹ cells/l, at thesame time as a confirmed or suspected infection.

Conditions (1) to (4) are known as the SIRS (Severe InflammatoryResponse Syndrome) criteria (Bone R C et al; Chest. 1992 June; 101(6):1644-55) and are a recognised international standard for diagnosis ofsevere inflammation. An individual exhibiting two or more of the SIRScriteria without a confirmed or suspected infection is classified ashaving non-infection associated SIRS.

The clinical definition of severe sepsis is sepsis as defined above,associated with sepsis-induced hypotension, organ dysfunction orperfusion abnormalities. Sepsis-induced hypotension is defined as asystolic blood pressure of <90 mm Hg or a reduction of <40 mm Hg frombaseline in the absence of other causes of hypotension. Perfusionabnormalities may include, but are not limited to hypoperfusion, lacticacidosis, oliguria, or an acute alteration in mental status. Severesepsis includes as a sub-category the condition of septic shock. Thiscondition is specifically defined by the presence of sepsis-inducedhypotension despite adequate fluid resuscitation along with the presenceof perfusion abnormalities. Individuals who are receiving inotropic orvasopressor agents may not be hypotensive at the time that perfusionabnormalities are measured.

For the treatment of severe sepsis, diagnosis prior to the onset of themore severe symptoms (hypotension, organ dysfunction or hypoperfusion)with the institution of adequate treatment is of the utmost importancefor a successful outcome (Rivers E et al. N Engl J Med 2001; 345(19):1368-77). For example, Kumar et al showed that mortality was correlatedto the number of hours passed after the onset of sepsis-inducedhypotension before the first treatment was given (Kumar et al. Crit CareMed 2006; 34(6):1589-96). A reliable biological or clinical marker todetermine as early as possible whether an individual is at risk ofdeveloping severe sepsis is needed to minimise the delay before theinstitution of treatment,

SUMMARY OF THE INVENTION

Heparin-binding protein (HBP, CAP37, Azurocidin) is a glycosylated,single chain, negatively, charged 37 kDa inactive serine proteasehomologue exhibiting 44% sequence identity with human neutrophilelastase. The three dimensional structure of HBP has been published(Iversen et al Nat Struct Biol. 1997 April; 4(4):265-8). It is containedin the azurofilic granulae of human neutrophils (Lindmark et al, JLeukoc Biol 1999; 66(4):634-43). It is a multifunctional protein thathas been shown to induce vascular leakage by altering the Ca²⁺ balanceof the blood vessel cytoskeleton (Gautam et al, Nature Medicine 2001;7(10):1123-7). The M-protein of group A streptococci (GAS) in complexwith fibrinogen has been shown to induce HBP release by stimulation ofthe B2-integrin receptor of neutrophils (Herwald et al, Cell 2004;116(3):367-79). LPS can also induce HBP release by an unknown mechanism(Rasmussen et al, FEBS Lett 1996; 390(1):109 12). The sequence of HBP ispublically available (for example as NCBI accession no. NP_(—)001691REGION: 27 . . . 248) and is reproduced below as SEQ ID NO.1

SEQ ID NO: 1IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVSTVVLGAYDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLPLQNATVRAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNNVCTGVLTRRGGICNGDGGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGP

The HBP levels in individuals exhibiting one or more of the SIRScriteria have not previously been investigated. The inventors have shownfor the first time that levels of HBP are increased in individuals whosubsequently develop severe sepsis. HBP levels are raised up to 12 hoursbefore sepsis-induced hypotension is recorded, but rapidly decrease iftherapy is instituted. The inventors have also shown for the first timethat the HBP/White Blood Cell count (WBC) ratio is elevated inindividuals who subsequently develop severe sepsis.

According to the invention there is thus provided a method ofidentifying whether or not an individual is at risk of developing severesepsis, which method comprises measuring HBP in the individual andthereby determining whether or not the individual is at risk ofdeveloping severe sepsis.

The method of the invention may further comprise measuring the WBC orneutrophil count (NC) in an individual, calculating the HBP/WBC ratio orHBP/NC ratio respectively, and thereby determining whether or not theindividual is at risk of developing severe sepsis.

The method of the invention may thus comprise measuring the HBP in anindividual and calculating the HBP/WBC level or HBP/NC ratio in anindividual, and thereby determining whether or not the individual is atrisk of developing severe sepsis.

The invention further provides:

-   -   an agent for the detection of HBP for use in determining whether        or not an individual is at risk of developing severe sepsis;    -   a test kit for use in a method for determining whether or not an        individual is at risk of developing severe sepsis, which test        kit comprises an agent for the detection of HBP in an        individual;    -   a method of reducing the risk of an individual developing severe        sepsis comprising:        -   (i) determining whether or not an individual is at risk of            developing severe sepsis using a method of the invention;            and        -   (ii) administering to an individual identified in (i) as at            risk, a therapeutically effective amount of an agent            suitable for the treatment of infection and/or intravenous            fluids.

DESCRIPTION OF THE FIGURES

FIG. 1 shows HBP concentration (FIG. 1 a), HBP/WBC ratios (FIG. 1 b),CRP levels (FIG. 1 c), IL-6 levels (FIG. 1 d) and lactate levels (FIG. 1e) in the severe sepsis group (n=51), sepsis group (n=95), infectionwithout SIRS group (n=44) and SIRS without infection group (n=12). Linewithin box: median; edges of box: quartiles (Q1, Q3); whiskers: range ofvalues; x and o: outlier values identified by patient number. Linesrepresenting cut-off values for HBP level of 20 ng/ml and HBP/WBC ratioof 2 for FIGS. 1 a and 1 b respectively

FIG. 2 shows an ROC curve for HBP level and/or HBP/WBC ratio, HBP level,HBP/WBC ratio, lactate, White blood cell count, CRP, and IL-6. Thestraight line from 0,0 to 1,1 is a reference line. Diagonal segments areproduced by ties.

FIG. 3 shows the HBP/WBC ratio (FIG. 3 a) or the HBP concentration (FIG.3 b) for each individual (n=51) in the severe sepsis group, plottedagainst the time from collection of the first plasma sample relative tothe time of the lowest measured blood pressure (indicated by the arrowat 0 hrs). Open circles in FIG. 3 a represent patients who fell belowthe HBP/WBC ratio cut off level but scored above the HBP concentrationcut off level. Open circles in FIG. 3 b represent patients who fellbelow the HBP concentration cut off level but scored above the HBP/WBCratio cut off level.

FIG. 4 shows the change in HBP/WBC ratio in consecutive plasma samplestaken from 16 patients with severe sepsis over 96 hours (FIGS. 4 a) and7 patients with sepsis, infection without SIRS or no infection over 72hours (FIG. 4 b). Each line represents an individual patient.

DETAILED DESCRIPTION OF THE INVENTION Diagnosis

The present invention relates to a method of identifying whether or nota subject is at risk of developing severe sepsis. The inventiontherefore relates to the diagnosis of susceptibility of an individual tosevere sepsis. The individual under test is typically suspected of beingat risk of developing severe sepsis. The individual is typically amammal. The mammal is typically a human or a domestic mammal such as ahorse, a cow, a sheep, a dog or a cat. The individual is preferably ahuman.

The individual may be suspected of being at risk of developing severesepsis because they have a confirmed or suspected infection, and/ordisplay one or more, or two or more, of the SIRS criteria. The SIRScriteria are:

(1) fever (temperature >38° C.) or hypothermia (temperature <36° C.);

(2) heart rate >90 beats per minute;

(3) respiratory rate >20 breaths per minute or PaCO₂<32 mm Hg; and

(4) white blood cell count >12(×10⁹ cells/L) or <4(×10⁹ cells/L).

The confirmed or suspected infection is typically one or more of abacterial, a parasitic or a fungal infection. A bacterial infection maybe caused by one or more Gram negative or Gram positive bacteria. Theone or more Gram negative bacteria may be selected from Escherichiacoli, Klebsiella spp. (typically K. pneumoniae or K. oxytoca),Enterobacter spp (typically E. cloacae or E. aerogenes), Bordetella spp.(typically B. bronchiseptica, B. pertussis or B. parapertussis),Chlamydia spp. (typically C. trachomatis), Legionella spp. (typically L.pneumophilia), Pseudemonas spp. (typically P. aeruginosa), Mycoplasmaspp. (typically M. pneumoniae), Haemophilus influenza, Serratiamarcescens, Proteus mirabilis, baumannii, Stenotrophomonas maltophiliaand Neisseria meningitidis (typically of serogroup A, B, C, H, I, K, L,X, Y, Z, 29E or W135). The one or more Gram positive bacteria may beselected from Staphylococcus spp. (typically S. aureus or Coagulasenegative Staphylococci), Streptococcus spp. (typically S. pneumoniae orS. pyogenes) and Enterococcus spp. (typically E. faecium or E.faecalis). A fungal infection may be caused by one or more fungiselected from Candida albicans, Candida tropicalis, Candidaparapsilosis, Candida krusei, Candida glabrata and Aperigillusfumigatus.

The confirmed or suspected infection may affect any part of the body.Typical examples include an infection which affects the lungs; therespiratory tract; the liver; the kidneys; the urinary tract; the skin(cutaneous and subcutaneous); the heart; the stomach; the intestines;the blood; the bones; the joints or any combination thereof. Theconfirmed or suspected infection may be meningitis.

The infection may be confirmed by diagnostic practices known in the art,for example microbial culture of samples taken from the individual,antigen testing of urine or other fluid samples taken from theindividual (especially for S. pneumoniae and Legionella sp. infections)or PCR analysis (especially for atypical pneumonia caused by bacterialinfection e.g. Mycoplasma, Legionella, Chlamydia. and B. pertussis).More recently multiplex PCR techniques have been developed which enablesimultaneous testing for multiple bacterial and fungal infections.

The infection may be suspected because of the presence of one or more ofthe following general symptoms: fever higher than 38° C.; chills; pain;an ache or tenderness; general feeling of tiredness; night sweats; and awound or incision with associated redness, heat, swelling or pain, orthat exudes a fluid that is white, yellowish or greenish.

The present invention may be used to confirm susceptibility in anindividual with one or more additional risk factors and/or one or morepredispositions toward the development of severe sepsis. Risk factorsthat increase susceptibility to developing severe sepsis typicallyinclude any factor which increases susceptibility to infection. Thesefactors can include a weakened immune system (i.e. the individual isimmuno-compromised), or the presence in a hospitalised patient of anintravenous line, surgical wound, surgical drain, or a site of skinbreakdown known as decubitus ulcers or bedsores. A diabetic individualis more prone to developing severe sepsis. The diagnostic method of theinvention may be carried out in conjunction with another assay orgenetic test to refine risk prediction.

Typically, the individual does not have a chronicinflammatory-associated disease and/or displays no symptoms which arespecifically associated with such diseases. Examples of such diseasesinclude atherosclerosis, Alzheimer's disease, asthma, rheumatoidarthritis, osteoarthritis, and inflammatory diseases of the bowel suchas Crohn's disease, Ulcerative colitis, Irritable bowel syndrome andInflammatory bowel disease. If the individual does have a chronicinflammatory-associated disease, they additionally have a confirmed orsuspected infection as defined above and/or display one or more, or twoor more, of the SIRS criteria.

Typically, the individual does not have severe sepsis or displays nosymptoms which would lead to a diagnosis of severe sepsis. Typicallysuch symptoms include sepsis-induced hypotension, organ dysfunction orperfusion abnormalities. Sepsis-induced hypotension is defined as asystolic blood pressure of <90 mm Hg or a reduction of <40 mm Hg frombaseline in the absence of other causes of hypotension. Perfusionabnormalities may include, but are not limited to hypoperfusion, lacticacidosis, oliguria, or an acute alteration in mental status. Severesepsis includes as a sub-category the condition of septic shock. Thiscondition is specifically defined by the presence of sepsis-inducedhypotension despite adequate fluid resuscitation along with the presenceof perfusion abnormalities. Individuals who are receiving inotropic orvasopressor agents may not be hypotensive at the time that perfusionabnormalities are measured.

The present invention involves measuring the level of HBP in anindividual. Typically the level of HBP is measured by determining theconcentration of HBP in a fluid sample taken from the individual.According to the present invention, an increased level of HBP comparedwith the baseline level or concentration indicates that the individualis susceptible to or at risk of developing severe sepsis. The baselinelevel is typically the level of HBP in an individual who is suspected ofbeing at risk of developing severe sepsis but does not subsequentlydevelop severe sepsis. For example the inventors have shown that, whenthe level of HBP is measured by determining the concentration of HBP ina plasma sample taken from an individual, individuals who developnon-severe sepsis have a median HBP concentration of about 8.5 ng/ml,individuals who have a confirmed or suspected infection but display oneor fewer SIRS criteria have a median HBP concentration of about 6.5ng/ml, and individuals who display of two or more SIRS criteria in theabsence of infection have a median HBP concentration of about 9 ng/ml.The median HBP concentration for all categories of individual who aresuspected of being at risk of developing severe sepsis but do notsubsequently develop severe sepsis is about 8 ng/ml.

In the present invention, in a fluid sample taken from an individual anincreased concentration of HBP associated with increased susceptibilityto or risk of developing severe sepsis is typically greater than about15 ng/ml, or greater than about 16, 17, 18, 19, 20, 21, 22, 23 or 24ng/ml. The increased concentration of HBP associated with increasedsusceptibility to or risk of developing severe sepsis is preferablygreater than about 20 ng/ml.

According to the present invention, the increase in HBP level orconcentration associated with increased susceptibility to or risk ofdeveloping severe sepsis is at least 2 fold, 2.5 fold or 3 fold relativeto the baseline level or concentration. The increase in HBP level orconcentration associated with increased susceptibility to or risk ofdeveloping severe sepsis is preferably at least 2.5 fold relative to thebaseline level or concentration.

The present invention may therefore also involve assessment of the ratioof HBP/white blood cell count (WBC) to determine the risk of developingsevere sepsis.

According to the present invention, an increased HBP/WBC ratio comparedwith the baseline ratio indicates that the individual is susceptible toor at risk of developing severe sepsis. The baseline ratio is typicallythe HBP/WBC ratio in an individual who is suspected of being at risk ofdeveloping severe sepsis but does not subsequently develop severesepsis. When the concentration of HBP in a fluid sample is measured inng/ml and the WBC in a blood sample is measured in number ofcells×10⁹/l, the inventors have shown that, for example, individuals whodevelop non-severe sepsis have a median HBP/WBC ratio of about 0.7:1,individuals who have a confirmed or suspected infection but display oneor fewer SIRS criteria have a median HBP/WBC ratio of about 0.85:1, andindividuals who display of two or more SIRS criteria in the absence ofinfection have a median HBP/WBC ratio of about 0.9:1. The median HBP/WBCratio for all categories of individual who are suspected of being atrisk of developing severe sepsis but do not subsequently develop severesepsis is about 0.75:1.

In the present invention, when the concentration of HBP in a fluidsample is measured in ng/ml and the WBC count in a blood sample ismeasured in number of cells×10⁹/l, an increased HBP/WBC ratio associatedwith increased susceptibility to or risk of developing severe sepsis istypically greater than about 1.4:1, or greater than about 1.5:1, 1.6:1,1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1 or 2.4:1. The increasedHBP/WBC ratio associated with increased susceptibility to or risk ofdeveloping severe sepsis is preferably greater than about 2.0:1.

According to the present invention, the increase in HBP/WBC ratioassociated with increased susceptibility to or risk of developing severesepsis is at least 1.5 fold, 2 fold, 2.5 fold or 3 fold relative to thebaseline ratio. The increase in HBP/WBC ratio associated with increasedsusceptibility to or risk of developing severe sepsis is preferably atleast 2.5 fold relative to the baseline ratio.

The present inventors have determined the mean neutrophil count (NC) inindividuals suspected of being at risk of developing severe sepsis isabout 80% of the white blood cell count (WBC). The present invention mayalso assess the ratio of HBP/NC to determine the risk of developingsevere sepsis.

According to the present invention, an increased HBP/NC ratio comparedwith the baseline ratio indicates that an individual is susceptible toor at risk of developing severe sepsis. The baseline ratio is typicallythe HBP/NC ratio in an individual who does not have an infection and/ordoes not display any of the SIRS criteria. When the concentration of HBPin a fluid sample is measured in ng/ml and the NC count in a bloodsample is measured in number of cells×10⁹/l, the inventors have shownthat, for example, individuals who develop non-severe sepsis have amedian HBP/NC ratio of about 0.55:1, individuals who have a confirmed orsuspected infection but display one or fewer SIRS criteria have a medianHBP/NC ratio of about 0.65:1, and individuals who display of two or moreSIRS criteria in the absence of infection have a median HBP/NC ratio ofabout 0.7:1. The median HBP/NC ratio for all categories of individualwho are suspected of being at risk of developing severe sepsis but donot subsequently develop severe sepsis is about 0.6:1.

In the present invention, when the concentration of HBP in a fluidsample is measured in ng/ml and the NC count in a blood sample ismeasured in number of cells×10⁹/l, an increased HBP/NC ratio associatedwith increased susceptibility to or risk of developing severe sepsis istypically greater than about 1.1:1, or greater than about 1.2:1, 1.3:1,1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1 or 1.9:1. The increased HBP/NC ratioassociated with increased susceptibility to or risk of developing severesepsis is preferably greater than about 1.6:1.

According to the present invention, the increase in HBP/NC ratioassociated with increased susceptibility to or risk of developing severesepsis is at least 1.5 fold, 2 fold, 2.5 fold or 3 fold relative to thebaseline ratio. The increase in HBP/NC ratio associated with increasedsusceptibility to or risk of developing severe sepsis is preferably atleast 2.5 fold relative to the baseline ratio.

The invention is typically carried out in vitro on a sample obtainedfrom the individual. The sample typically comprises a body fluid of theindividual. The sample is preferably a blood, plasma, serum, mine,cerebrospinal fluid or joint fluid sample. The sample is most preferablya blood sample. The sample is typically processed prior to beingassayed, for example by centrifugation. The sample may also be typicallystored prior to assay, preferably below −70° C.

Standard methods known in the art may be used to assay the level of HBP.These methods typically involve using an agent for the detection of HBP.The agent typically binds specifically to HBP. The agent may be anantibody specific for HBP. By specific, it will be understood that theagent or antibody binds to HBP with no significant cross-reactivity toany other molecule, particularly any other protein. For example, anagent or antibody specific for HBP will show no significantcross-reactivity with human neutrophil elastase. Cross-reactivity may beassessed by any suitable method.

An antibody used in the method of the invention may either be a wholeantibody or a fragment thereof which is capable of binding to HBP. Theantibody may be monoclonal. Such a whole antibody is typically anantibody which is produced by any suitable method known in the art. Forexample, polyclonal antibodies may be obtained by immunising a mammal,typically a rabbit or a mouse, with HBP under suitable conditions andisolating antibody molecules from, for example, the serum of saidmammal. Monoclonal antibodies may be obtained by hybridoma orrecombinant methods.

Hybridoma methods involve immunising a mammal, typically a rabbit or amouse, with HBP under suitable conditions, then harvesting the spleencells of said mammal and fusing them with myeloma cells. The mixture offused cells is then diluted and clones are grown from single parentcells. The antibodies secreted by the different clones are then testedfor their ability to bind to HBP, and the most productive and stableclone is then grown in culture medium to a high volume. The secretedantibody is collected and purified.

Recombinant methods involve the cloning into phage or yeast of differentimmunoglobulin gene segments to create libraries of antibodies withslightly different amino acid sequences. Those sequences which give riseto antibodies which bind to HBP may be selected and the sequences clonedinto, for example, a bacterial cell line, for production.

Typically the antibody is a mammalian antibody, such as a primate,human, rodent (e.g. mouse or rat), rabbit, ovine, porcine, equine orcamel antibody. The antibody may be a camelid antibody or sharkantibody. The antibody may be a nanobody. The antibody can be any classor isotype of antibody, for example IgM, but is preferably IgG.

The fragment of whole antibody that can be used in the method comprisesan antigen binding site, e.g. Fab or F(ab)2 fragments. The wholeantibody or fragment may be associated with other moieties, such aslinkers which may be used to join together 2 or more fragments orantibodies. Such linkers may be chemical linkers or can be present inthe form of a fusion protein with the fragment or whole antibody. Thelinkers may thus be used to join together whole antibodies or fragmentswhich have the same or different binding specificities, e.g. that canbind the same or different polymorphisms. The antibody may be abispecific antibody which is able to bind to two different antigens,typically any two of the polymorphisms mentioned herein. The antibodymay be a ‘diabody’ formed by joining two variable domains back to back.In the case where the antibodies used in the method are present in anyof the above forms which have different antigen binding sites ofdifferent specificities then these different specificities are typicallyto polymorphisms at different positions or on different proteins. In oneembodiment the antibody is a chimeric antibody comprising sequence fromdifferent natural antibodies, for example a humanised antibody.

Methods to assess HBP level typically involve contacting a sample withan agent or antibody capable of binding specifically to HBP. Suchmethods may include dipstick assays and Enzyme-linked ImmunosorbantAssay (ELISA). Typically dipsticks comprise one or more antibodies orproteins that specifically bind HBP. If more than one antibody ispresent, the antibodies preferably have different non-overlappingdeterminants such that they may bind to HBP simultaneously.

ELISA is a heterogeneous, solid phase assay that requires the separationof reagents. ELISA is typically carried out using the sandwich techniqueor the competitive technique. The sandwich technique requires twoantibodies. The first specifically binds HBP and is bound to a solidsupport. The second antibody is bound to a marker, typically an enzymeconjugate. A substrate for the enzyme is used to quantify theHBP-antibody complex and hence the amount of HBP in a sample. Theantigen competitive inhibition assay also typically requires anHBP-specific antibody bound to a support. An HBP-enzyme conjugate isadded to the sample (containing HBP) to be assayed. Competitiveinhibition between the HBP-enzyme conjugate and unlabeled HBP allowsquantification of the amount of HBP in a sample. The solid supports forELISA reactions preferably contain wells.

The present invention may also employ methods of measuring HBP that donot comprise antibodies. High Performance Liquid Chromatography (HPLC)separation and fluorescence detection is preferably used as a method ofdetermining the HBP level. HPLC apparatus and methods as describedpreviously may be used (Tsikas D et al. J Chromatogr B Biomed Sci Appl1998; 705: 174-6) Separation during HPLC is typically carried out on thebasis of size or charge. Prior to HPLC, endogenous amino acids and aninternal standard L-homoarginine are typically added to assay samplesand these are phase extracted on CBA cartridges (Varian, Harbor City,Calif.). Amino acids within the samples are preferably derivatized witho-phthalaldehyde (OPA). The accuracy and precision of the assay ispreferably determined within quality control samples for all aminoacids.

Standard methods known in the art may be used to measure the white bloodcell count or neutrophil count in an individual. Such methods includedautomated or manual counting.

The invention further provides a diagnostic kit that comprises means formeasuring the HBP level in an individual and thereby determining whetheror not the individual is at risk of developing severe sepsis. The kittypically contains one or more antibodies that specifically bind HBP.For example, the kit may comprise a monoclonal antibody, a polyclonalantibody, a single chain antibody, a chimeric antibody, a CDR-graftedantibody or a humanized antibody. The antibody may be an intactimmunoglobulin molecule or a fragment thereof such as a Fab, F(ab′)₂ orFv fragment. If more than one antibody is present, the antibodiespreferably have different non-overlapping determinants such that theymay bind to HBP simultaneously.

The kit may additionally comprise means for the measurement of the WBCcount in an individual.

The kit may additionally comprise one or more other reagents orinstruments which enable any of the embodiments of the method mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toisolate HBP from sample, means to obtain a sample from the individual(such as a vessel or an instrument comprising a needle) or a supportcomprising wells on which quantitative reactions can be done. The kitmay, optionally, comprise instructions to enable the kit to be used inthe method of the invention or details regarding which individuals themethod may be carried out upon.

Therapy

The present invention also relates to the treatment of an individualidentified by a method of the invention as at risk of developing severesepsis. Thus, a substance for use in reducing the risk of developingsevere sepsis may be used in the manufacture of a medicament for use inthe treatment of an individual identified by a method of the inventionas at risk of developing severe sepsis. The condition of an individualidentified by a method of the invention as at risk of developing severesepsis can therefore be improved by administration of such a substance.Severe sepsis can thus be prevented. A therapeutically effective amountof a substance useful for reducing the risk of developing severe sepsismay be given to an individual identified by a method of the invention asin need thereof. Substances suitable for reducing the risk of developingsevere sepsis typically include one or more antibiotics and/or one ormore intravenous fluids. The one or more antibiotics are typically broadspectrum antibiotics. The broad spectrum antibiotics are typicallyselected from one or more aminoglycosides, cephalosporins,fluoroquinolones, lincosamides, macrolides, penicillins, sulfonamides,or tetracyclins. For example, suitable antibiotics include, but are notlimited to, Gentamicin, Kanamycin, Neomycin, Streptomycin, Tobramycin,Cefazolin, Cephalexin, Cephapirin, Cephradine, Cefuroxime, Cefixime,Cefotaxime, Ceftazidime, Ceftizoxime, Ceftriaxone, Ciprofloxacin,Levofloxacin, Ofloxacin, Clindamycin, Azithromycin, Clarithromycin,Erythromycin, Amoxicillin, Ampicillin, Ampicillin-Sulbactam,Cloxacillin, Dicloxacillin, Mezlocillin, Nafcillin, Oxacillin,Penicillin G Benzathine, Penicillin G Potassium, Penicillin G Procaine,Penicillin V Potassium, Piperacillin, Ticarcillin,Ticarcillin-Clavulanate potassium, Pyrimethamine-Sulfadoxine,Sulfadizine, Sulfisoxazole, Sulfmethoxazole,Trimethoprim-sulfamethoxazole, Chlortetracycline, Doxycycline, andTetracycline.

A substance useful for reducing the risk of developing severe sepsisaccording to the invention is typically formulated for administration inthe present invention with a pharmaceutically acceptable carrier ordiluent. The pharmaceutical carrier or diluent may be, for example, anisotonic solution. For example, solid oral forms may contain, togetherwith the active substance, diluents, e.g. lactose, dextrose, saccharose,cellulose, corn starch or potato starch; lubricants, e.g. silica, talc,stearic acid, magnesium or calcium stearate, and/or polyethyleneglycols; binding agents; e.g. starches, gum arabic, gelatin,methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone;disaggregating agents, e.g. starch, alginic acid, alginates or sodiumstarch glycolate; effervescing mixtures; dyestuffs; sweeteners; wettingagents, such as lecithin, polysorbates, laurylsulphates; and, ingeneral, non-toxic and pharmacologically inactive substances used inpharmaceutical formulations. Such pharmaceutical preparations may bemanufactured in known manner, for example, by means of mixing,granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions orsuspensions. The syrups may contain as carriers, for example, saccharoseor saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together with theactive substance, a pharmaceutically acceptable carrier, e.g. sterilewater, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and ifdesired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain ascarrier, for example, sterile water or preferably they may be in theform of sterile, aqueous, isotonic saline solutions.

A therapeutically effective amount of a substance used in the preventionof severe sepsis is administered to a patient identified according to amethod of the invention. The dose, for example of an antibiotic, may bedetermined according to various parameters, especially according to thesubstance used; the age, weight and condition of the patient to betreated; the route of administration; and the required regimen. Again, aphysician will be able to determine the required route of administrationand dosage for any particular patient. A typical daily dose is fromabout 0.1 to 50 mg per kg of body weight, according to the activity ofthe specific antibiotic, the age, weight and conditions of the subjectto be treated and the frequency and route of administration. Preferably,daily dosage levels are from 5 mg to 2 g. That dose may be provided as asingle dose or may be provided as multiple doses, for example taken atregular intervals, for example 2, 3 or 4′ doses administered daily.

The following Example illustrates the invention:

EXAMPLE 1. Methods Study Participants

202 adult patients with clinically suspected infection were enrolled ina prospective study at the Infectious Disease Clinic, Lund UniversityHospital, Sweden, between March 2006 and April 2007. Inclusion criteriawere fever >38° C. and antibiotic treatment for less than 24 hours.Duration of antibiotic treatment, demographics, SIRS criteria andsystolic blood pressure (SBP) were recorded at admittance. C-reactiveprotein (CRP), lactate and WBC count were analysed, and plasma samplesfor later analysis of HBP, IL-6 levels were obtained within 12 hoursfrom admission. In 20 patients serial plasma samples were obtained forup to 96 hours in parallel with recording SIRS criteria and SBP. Afterdischarge, the final diagnosis and 28 d mortality was recorded and thepatients were classified according to the SIRS criteria (Bone et al,Chest 1992; 101(6): 1644-55).

Severe sepsis (group 1) was defined as the presence of sepsis and SBP<90 mmHg or a >40 mmHg SBP fall from baseline within 24 h of collectionof the blood samples, sepsis (group 2) was defined as display of two ormore SIRS criteria together with an infection, non-sepsis (group 3) wasdefined as display of one SIRS criterion together with an infection, andnon-infection (group 4) was defined as display of two or more SIRScriteria together with a non-infectious final diagnosis.

Final infection diagnoses were pneumonia (n=61), upper respiratory tractinfection (n=35), urinary tract infection (n=38), cutaneous andsubcutaneous infection (n=29), endocarditis (n=4), gastroenteritis(n=12), or other infections including tropical infections (n=11). In 35patients (17%) bacteremia was diagnosed (17 Gram-negative and 18Gram-positive bacteria). Non-infectious diagnoses with display of two ormore SIRS criteria were pulmonary embolis and systemic vasculitis).Blood samples were collected in 4 mL plastic plasma citrate tubes,immediately centrifuged at 3000 rpm for 10 min, alloquated and stored at−70° C. until analysis.

Analysis of HBP, IL-6, CRP, Lactate, WBC and HBP/WBC Ratio

The concentration of HBP (ng/ml) was determined by a sandwich ELISA asdescribed in (Tapper et al; Blood 2002; 99(5): 1785-93). Plasma sampleswere diluted 1/40 in PBS and run in duplicates. Recombinant human HBPwas produced and purified as described in (Rasmussen et al; FEBS Lett1996; 390(1):109-12). Mouse monoclonal antibodies (2F23A) and rabbitantiserum (409A) to recombinant HBP were prepared and purified asdescribed in (Lindmark et al; J. Leukoc. Biol 1999; 66(4):634-43) andused at 1/3000 and 1/7000 respectively. Peroxidase-conjugated goatantirabbit IgG from Bio-Rad Laboratories (Richmond, Calif.), were usedat 1/3000. A HBP/WBC ratio was calculated by dividing the HBPconcentration (ng/ml) with the WBC (×10⁹ cells/L).

IL-6 was measured with a commercial human IL-6 kit (Quantikine, R&DSystems, UK), detection limit 3 pg/mL. Plasma samples were diluted 1/40.CRP and lactate analyses were performed on a Roche Hitachi Modular-Pwith reagents from Roche Diagnostics (Mannheim, Germany) according tothe manufacturer's description with the exception that the samples forthe lactate analysis were taken from plasma citrate tubes instead ofoxalate-flouride tubes.

White blood cell count (WBC) was measured in a Sysmex XE2100 accordingto the manufacturer's description (Sysmex).

Statistical Analysis

Data are presented as medians, interquartile ranges. Significancetesting was carried out using the Mann-Whitney rank sum test. Atwo-tailed p-value <0.05 was considered statistically significant.Receiver-operating characteristic (ROC) curves (DeLong et al; Biometrics1988; 44(3): 837-45) and the area under the curve (AUC) were determinedfor HBP, HBP/WBC ratio, CRP, WBC and IL-6. AUC values are reported withthe 95% confidence interval (95% CI). Sensitivities, specificities,positive predictive values and negative predictive values werecalculated from cross-tabulations. The positive likelihood ratio andnegative likelihood ratio are also reported in Table 1.

2. Results Characteristics of the Study Participants

202 patients who met the inclusion criteria were included. The patientswere classified into the 4 following groups: 51 patients with severesepsis (group 1), 95 patients with sepsis (group 2), 44 patients withnon-sepsis (group 3) and 12 patients with non-infection (group 4),Median age and male/female ratio of the 4 groups were 62 y; 31/20, 57 y;42/53, 44 y; 15/29, and 73 y; 11/1 respectively. Bacteremia was found in22, 11, and 2 patients in group 1, 2, and 3 respectively.

Analysis of HBP Levels and HBP/WBC Ratios in Study Participants

At admission, HBP levels were significantly higher in the severe sepsisgroup (p<0.0001) (FIG. 1 a), with 42/51 patients exceeding a cut-offlevel of 20 ng/ml, as compared to 1/95, 0/44 and 0/12 patients in group2, group 3 and group 4, respectively. Also, patients in the severesepsis group showed significantly (p <0.0001) higher HBP/WBC ratios ascompared to the other groups (FIG. 1 b). A HBP/WBC ratio above 2.0 wasfound in 44/51 patients in the severe sepsis group, 2/95 patients in thesepsis group, 0/44 patients in the non-sepsis group and 0/12 patients inthe non-infection group. Moreover, in the severe sepsis group 47/51patients had either a plasma HBP level >20 ng/ml or a HBP/WBCratio >2.0, as compared to 2/95 patients in the sepsis group and 0patients in the 2 other groups (Table 1).

Similarly, CRP, IL-6 and lactate levels were also significantly higher(p=0.003, p<0.0001, p<0.0001) in the severe sepsis group although therewas a considerable overlapping between groups (FIG. 1 c-e).

Analysis of Predictive Value of HBP Level and HBP/WBC Ratio

Assuming a 25% prevalence of severe sepsis, as in the present study, thefollowing variables showed better specificity, sensitivity, positivepredictive value (PPV), negative predictive value (NPV), positivelikelihood ratio (PLR) and negative likelihood ratio (NLR) in diagnosingsevere sepsis as compared to all different cut off levels of CRP andIL-6 calculated:

-   -   a HBP level >20 ng/ml indicates individual will develop severe        sepsis; or    -   a HBP/WBC ratio >2.0 indicates individual will develop severe        sepsis; or    -   either a HBP level >20 ng/ml or a HBP/WBC ratio >2.0 indicates        individual will develop severe sepsis

These findings are summarised in Tables 1a, 1b and 1c, and are supportedby the ROC curve (FIG. 2). Statistical analysis of the ROC curve isshown in table 2.

HBP Level and HBP/WBC Ratio Predict Severe Sepsis Before Onset ofClinical Symptoms

In 20 of the 51 patients in the severe sepsis group the HBP value waselevated up to 12 h before the lowest SBP was reached (FIGS. 3 a and 3b). The HBP levels rapidly decreased within 24 h in the 11 patients whowere treated with adequate antibiotics and intravenous fluids and whorecovered without complications. In 5 cases where the patients continuedto be circulatory unstable the HBP levels, however, remained elevated, 1of these patient died within 28 days of the inclusion date (FIG. 4 a).

In all 5 patients died, all in the severe sepsis group and all within1-4 days after the last collected elevated HBP sample. The HBP levelsremained low below 20 ng/ml in 6 patients with sepsis and 1 patient withnon-sepsis who were followed with serial sampling (FIG. 4 b).

20 patients in the severe sepsis group had increased HBP levels orHBP/WBC ratios at admission but normal SBP, indicating that theseindicators are able to predict severe sepsis before standard clinicaldiagnosis is possible. This is exemplified by a 70-year-old womanpresenting with a 3-day history of fever, diarrhoea and vomiting. Thephysical examination was unremarkable except for fever 38° C., pulserate 100 but normal SBP of 130 mm/Hg, and she was hospitalized with atentative diagnosis of gastroenteritis. She was given 1000 ml i.v.crystalloid fluid. Six hours later she developed severe hypotension withSBP 70 mm/Hg, respiratory distress and was immediately transferred tothe ICU, where ARDS (Adult Respiratory Distress Syndrome) withdisseminated intravascular coagulation due to E.coli septicemia waslater diagnosed. At admission her HBP level was 80 ng/ml and HBP/WBCratio was 4.2.

TABLE 1a Sensitivity, specificity, positive predictive value, negativepredictive value, positive likelihood ratio and negative likelihoodratio of tested variables in diagnosing severe bacterial sepsis VariableCut-off Sensitivity % Specificity % PPV % NPV % PLR NLR HBP (ng/ml) >2082.4 99.3 97.7 94.3 118 0.18 HBP/WBC >2 86.3 98.7 95.6 95.5 66.4 0.14ratio HBP/WBC >2 92.2 98.7 95.9 97.4 71 0.08 ratio or or HBP level >20(ng/ml) CRP (mg/l) >50 88.2 36.4 31.9 90.2 1.39 0.32 >100 82.3 53.6 37.590.0 1.77 0.33 >150 64.7 71.3 42.9 85.6 2.25 0.49 >200 37.2 80.8 39.679.2 1.94 0.77 IL6 (pg/ml) >100 78.4 72.8 49.4 91.0 2.88 0.30 >200 62.777.5 48.5 86.0 2.78 0.48 >500 47.0 84.0 50.0 82.5 2.94 0.63 >1000 43.090.0 59.5 82.4 4.30 0.23 Lactate(mmol/l) >2.5 27.5 98.0 82.4 79.6 13.70.74 >2.0 41.2 91.8 63.6 81.8 5.0 0.64 >1.5 52.9 77.6 45.0 82.6 2.40.61 >1 88.2 46.3 36.3 91.9 1.6 0.25

TABLE 1b Sensitivity and specificity of different cut off levels of HBPin diagnosing severe bacterial sepsis HBP cut off (ng/ml) SpecificitySensitivity >15 95.1 88.2 >16 98.0 88.2 >17 98.6 84.3 >19 98.6 84.3 >2099.3 82.4 >21 99.3 80.4 >22 99.3 78.4 >23 99.3 76.5 >24 99.3 74.5

TABLE 1c Sensitivity and specificity of different cut off levels ofHBP/WBC ratio in diagnosing severe bacterial sepsis HBP/WBC ratio cutoff Specificity Sensitivity 1.4 87.1 92.2 1.5 95.0 88.2 1.7 95.5 86.31.8 96.3 86.3 1.9 98.0 86.3 2.0 98.6 86.3 2.1 98.6 84.3 2.2 98.6 81.42.3 98.6 78.4 2.4 98.6 74.5 3.6 99.3 54.9

TABLE 2 Analysis of ROC curve for tested variables in diagnosing severebacterial sepsis Asymptotic 95% Area Std. Asymp- Confidence IntervalTest Result Under Error totic Lower Upper Variable(s) Curve (a) Sig. (b)Bound Bound HBP (ng/ml) .954 .017 .000 .920 .988 HBP/WBC ratio .949 .022.000 .905 .993 HBP/WBC ratio .960 .019 .000 .923 .997 or HBP level(ng/ml) CRP (mg/ml) .719 .040 .000 .641 .797 IL6 (pg/ml) .789 .038 .000.714 .863 Lactate(mmol/l) .769 .050 .000 .697 .841 WBC count .511 .050.820 .413 .608

1-15. (canceled)
 16. An agent for the detection of HBP for use indetermining whether or not an individual is at risk of developing severesepsis.
 17. An agent according to claim 16 wherein the agent comprisesan HBP-specific antibody. 18-21. (canceled)
 22. A test kit for use in amethod for determining whether or not an individual is at risk ofdeveloping severe sepsis, which test kit comprises an agent for thedetection of HBP in an individual.
 23. The kit of claim 21 additionallycomprising means for the measurement of the WBC in an individual.